Funding from the Australian Research Council (ARC) and National Health and Medical Research Council (NHMRC) is gratefully acknowledged.

The authors have nothing to disclose.

El modelo de rata mesentérica linfáticos canulación permite la cuantificación directa de la concentración y la velocidad de transporte de varias células y moléculas (tales como lípidos y fármacos) del intestino a la linfa y los cambios a estos que se producen en respuesta al desafío de diversas sustancias (dieta , antígenos, fármacos, formulaciones, etc.) 10,27 y la enfermedad (cáncer, los virus, la colitis, la resistencia a la insulina, etc.) 5-7. Los componentes rec...

La linfa fluye desde el intestino delgado a través de un proceso unidireccional que se origina en lactíferos individuales que están contenidos dentro de cada pequeño vellosidades intestinales 1. Lacteales son relativamente permeable al fluido, macromoléculas y células y la formación de la linfa por lo tanto comienza con la entrada de estos factores en lacteales. La linfa inicial en los lacteales fluye posteriormente desde el intestino a través de una red de microvasos linfáticos, la recolección (aferente) los vasos linfáticos, una serie de ganglios linfáticos mesentéricos y en última instancia los post-ganglionar (eferente) los vasos linfáticos. Dentro de los nodos, la linfa pasa a través de una serie de senos medulares donde se produce el intercambio con las células inmunes residentes nodo, así como material que entran en el nodo de la sangre. Todos los ganglios que fluye desde el intestino delgado finalmente converge en el conducto linfático eferente mesentérica superior y, posteriormente, la cisterna del quilo. La cisterna del quilo además contiene linfático de drenaje de los tejidos periféricos caudales, intestinal, regiones hepáticas y lumbares y se une al conducto linfático torácico junto con la linfa del mediastino y partes craneales del cuerpo. El conducto linfático torácico vacía linfático directamente en el sistema venoso en la unión de las venas yugular interna y subclavia izquierda. El protocolo descrito aquí, que permite la recogida de la linfa directamente desde el conducto linfático mesentérico superior, por lo tanto facilita el análisis de diversos factores que transitan directamente desde el intestino a la circulación (general) sistémica a través del sistema linfático intestinal. Las principales funciones fisiológicas asignados al sistema linfático intestinal son para mantener el equilibrio de fluidos, para facilitar la absorción de los lípidos y la molécula lipófila, y para permitir respuestas inmunes apropiadas 1. Las células tumorales y virus también se propagan a través de los vasos linfáticos intestinales 2-4 y principales cambios se producen dentro de los vasos linfáticos en diversas patologías inflamatorias y metabólicas 5-7. Latanulation del conducto linfático mesentérico para recoger la linfa en el mesenterio permite un análisis de flujo de fluido mayor a través de los vasos linfáticos intestinales, así como la cuantificación de la tasa de concentración y el transporte de varias células y moléculas. Los cambios en la concentración o el tránsito de estos factores en respuesta a diversos desafíos (por ejemplo, dietas, antígenos, drogas) y en modelos de enfermedad (por ejemplo, colitis, VIH, diabetes) pueden también ser evaluados. Si bien es imposible describir ampliamente cada componente linfático que puede ser analizada y comparada aquí, linfático mesentérico consiste simplista de acuosa, lípidos y las fases celulares. Componentes de interés en la fase acuosa incluyen péptidos y proteínas tales como antígenos o tolerógenos 8, mensajeros inmunes tales como citoquinas y mediadores de mastocitos, 9 y mediadores metabólicos tales como incretinas 10. La fracción celular de la linfa mesentérica post-nodal consiste casi completamente (más del 99%) de lymphocytes 11. Varias células inmunes (células dendríticas, mastocitos, etc.) entran en los vasos linfáticos mesentéricos pre-nodales pero permanecen dentro del nodo 12. Si las células dentro de la linfa aferente son de interés, es posible recoger estas células a través de la eliminación de la nódulos linfáticos mesentéricos varios días antes de la canulación del conducto linfático mesentérico 12. De esta manera los aferentes y eferentes conductos linfáticos están directamente conectados y las células en los ganglios linfáticos aferentes pasan directamente en el conducto linfático mesentérico. El tránsito y el fenotipo de varias células inmunes que pasan a través de los vasos linfáticos intestinales por lo tanto pueden ser examinados. Quizás la razón más común citada para la recogida de la linfa mesentérica hasta la fecha, sin embargo, es estudiar el procesamiento intestinal, la absorción y el transporte de lípidos de la dieta y las moléculas lipofílicas 10. Después de la ingestión, lípidos de la dieta son digeridos (por ejemplo, de triglicéridos de ácidos grasos y monoglicéridos, phospholipid a los ácidos grasos y lisofosfolípidos, colesterol y éster de ácido graso y colesterol, etc.) y dispersas dentro de la luz intestinal en pequeñas micela y las estructuras vesiculares por medio de la adición de anfífilos de la bilis (fosfolípidos, colesterol y sales biliares) y la acción de enzimas pancreáticas 10,13. De aquí que se absorben en los enterocitos. Una proporción de los componentes absorbidos se re-esterificados para formar triglicéridos, fosfolípidos y ésteres de colesterol dentro de las células absorbentes (enterocitos). Estas re-esterificados lípidos se ensamblan a partir de una combinación de componentes lipídicos exógena ingerida y componentes de lípidos endógenos de la bilis secretada, piscinas de lípidos de la mucosa o el suministro de sangre intestinal 13. Desde aquí los lípidos esterificados se almacenan ya sea dentro de los enterocitos o ensamblados en lipoproteínas intestinales (quilomicrones, lipoproteínas de muy baja densidad (VLDL)), junto con diversos apoproteínas y otras moléculas lipófilas ( <em&gt; por ejemplo, vitaminas) 10,13. Después de salir de los enterocitos, lipoproteínas son transportados específicamente desde el intestino a la circulación sistémica a través del sistema linfático mesentérico como los lactíferos intestinales son más permeables a su entrada de los capilares sanguíneos intestinales. Una proporción de los componentes de lípidos absorbidos también son transportados desde el intestino a la circulación sistémica a través de los capilares sanguíneos y la vena porta como individuales, no de lipoproteínas asociadas, las moléculas 14. En general, sin embargo, la ruta de transporte de la vena porta es sólo un jugador significativo en la absorción de los lípidos de longitud de cadena corta y media. La recogida de la linfa mesentérica por lo tanto permite la evaluación del transporte de lipoproteínas y componentes asociados (lípidos, moléculas lipófilas, APO-proteínas) desde el intestino. Las lipoproteínas se pueden cuantificar y caracterizados con la ventaja de que las lipoproteínas ganglios mesentéricos, en general, están en una nascent estado, ya que no han sido ampliamente modificado por enzimas sistémicas tales como la lipoproteína lipasa 15. Mientras que el modelo de rata canulada linfático mesentérico ha históricamente quizás ha descrito más ampliamente para el análisis de transporte de lípidos / lipoproteínas de desde el intestino, un área de interés es la ampliación de la función de los vasos linfáticos en el transporte de fármacos lipófilos, profármacos y otros xenobióticos 13,16 que es el foco del modelo descrito aquí. Drogas lipofílicas (generalmente aquellos con log P&gt; 5 y solubilidad en cadena larga de triglicéridos&gt; 50 mg / g, aunque excepciones son evidentes) 17,18, profármacos 19 y otros xenobióticos 13,16 puede tener acceso a los linfáticos intestinales de forma pasiva o por integrando activamente en las vías de transporte de lipoproteínas intestinal 19. Así, la técnica de canulación linfático mesentérico de rata tiene muchas aplicaciones. Bollman et al. Descrita por primera vez un technique para canular el conducto linfático mesentérico en ratas en 1948 20. Desde entonces un número de variaciones sobre el modelo se han descrito. Por ejemplo, la colección se puede producir cuando la rata se anestesió con varios anestésicos 21,22, o en el estado consciente mientras refrenado 15 o se mueve libremente 23,24. Las ratas se pueden administrar diferentes soluciones de rehidratación y otras sustancias tales como lípidos y formulaciones de fármacos a diferentes velocidades en el estómago, intestino o parenteral (típicamente 0-5 ml / hr) 25. En algunos estudios, el conducto linfático torácico en lugar de conducto linfático mesentérico se canula para estimar el transporte desde el intestino a través de los vasos linfáticos aunque esto puede sobreestimar el tránsito en el intestino delgado, en función del factor de interés, ya que el conducto linfático torácico también recibe la linfa de otra regiones 22,26. Modelos de canulación linfáticos también se han descrito en varias otras especies, incluyendo ratones 15,27, mini-pigs 12, 28,29 ovejas, cerdos y perros 30 31. Sin embargo, el modelo de rata es la más amplia y coherente citado. Los protocolos detallados para la canulación del conducto linfático mesentérico seguida de la recogida de la linfa en consciente 25 o anestesiado 22 ratas y ratones 15,27 se han publicado previamente y el lector interesado se dirige a estos protocolos. Este protocolo es el primero en demostrar la técnica en un formato visualizado. El modelo de rata linfático canulado tiene ventajas sobre los modelos animales más grandes en términos de costo, la facilidad de la cirugía y las consideraciones éticas. Cuando se compara con el modelo de ratón, la cirugía canulación linfático mesentérico es también más fácil en la rata Aunque el modelo de ratón permite estudios más detallados en los animales transgénicos 27. Sin embargo, hay algunas limitaciones del modelo de rata, en particular aquellos asociados con diferencias en la fisiología, que extrapolat límiteiones a otras situaciones pre-clínicos y clínicos. Por ejemplo, en el flujo de bilis de rata es constante e independiente de la ingesta de alimentos mientras que en mayor alimentos especie o lípidos estimulan el flujo de bilis 32. Esto crea desafíos para la obtención de los entornos de pre y post-prandial de representación en la rata que reflejan lo que se ve en las especies y los seres humanos más grandes. Para los estudios de administración de fármacos, las especies más grandes también pueden ser preferidos cuando se evalúa el transporte linfático después de la administración de la dosis humana realista forma 25. En un estudio reciente, se encontró que las tasas de transporte de lípidos en la linfa mesentérica ser comparables entre especies (ratón, rata, perro) después de la administración de una masa equivalente y el tipo de lípidos que proporciona cierta confianza en la extrapolación de los datos de transporte de lípidos a través de especies 27. Sin embargo, el transporte de un modelo lipofílica drogas, halofantrina, clasificados en orden de tamaño de los animales (es decir, perro&gt; rata&gt; ratón). Un factor de escala puede, pues, ser obligado a exextrapolar los datos de transporte de drogas linfáticos de rata para otras especies. Una limitación de los modelos de canulación de la linfa, en general, es que la recogida pasiva linfático directamente de un conducto linfático puede modificar el flujo y transporte linfático desde los vasos linfáticos trabajan en contra de un gradiente de presión que se altera una vez que el recipiente se canula 33. El modelo canulación linfáticos también puede ser difícil de establecer en los laboratorios que no están familiarizados con la técnica. Así, se han descrito modelos alternativos. Por ejemplo, el tránsito de factores través del sistema linfático intestinal, tales como lipoproteínas y moléculas lipofílicas, se ha estudiado indirectamente a través de la colección de la sangre. Uno de estos modelos consiste en comparar las concentraciones sanguíneas de lípidos y / o drogas tras la administración oral en presencia y ausencia de inhibidores (por ejemplo, colchicina, Pluronic L81, cicloheximida) de la producción de lipoproteínas intestinal que bloquean el transporte linfático 34. Una ventajade los modelos que cuantifican el transporte linfático indirectamente a través de la recolección de muestras de sangre es que permite cierta evaluación del transporte linfático en los seres humanos como no se requiere cirugía invasiva 35. Sin embargo, los inhibidores de transporte linfático no son específicos y los factores que son transportados a través de los vasos linfáticos se diluyen y se modifican en la circulación sistémica que complica tales evaluaciones. In vitro alternativas también se han descrito. Por ejemplo, los cultivos de células Caco-2 o aislado de los enterocitos se han utilizado para estudiar en más detalle la secreción intestinal de moléculas que entran en los vasos linfáticos 36-38. Un modelo avanzado in vitro que es más representativo del microambiente intestinal humano también fue recientemente descrito 39. En este modelo de una capa de células endoteliales linfáticas es co-cultivadas con células Caco-2 que permite análisis más detallado de la transferencia de sustancias desde el intestino en los vasos linfáticos. Sin embargo, en vitro sistemas de células carecen de flujo de intercambio y transferencia denominado interconexión con un lumen intestinal y sangre subyacente y de aporte vascular linfático. En un enfoque alternativo, Kassis et al. Estableció un doble canal (vídeo de alta velocidad de campo claro y fluorescencia) en el sistema de formación de imágenes in situ que permite comparaciones cuantitativas entre contracción de los vasos, el flujo de la linfa y las concentraciones de lípidos fluorescentes en los vasos linfáticos mesentéricos 33. Una ventaja de este modelo sobre el mencionado en sistemas in vitro es que permite un seguimiento preciso del paso de las células inmunes a través de los vasos linfáticos. Las medidas absolutas de lípidos masa (o drogas) de transporte son, sin embargo, aún no se establecerán a partir de los métodos de imagen. In vitro e in silico enfoques para predecir específicamente el grado de transporte del fármaco lipofílico a través de los vasos linfáticos intestinales también se han publicado 40-42. Por ejemplo, la afinidad ex vivo de varios ccomuestos para quilomicrones plasma se encontró correlación razonablemente bien con su transporte linfático en vivo 41. Posteriormente, el mismo grupo estableció un modelo en silico para predecir la afinidad fármaco para quilomicrones basados ​​en múltiples propiedades fisicoquímicas 40. Holm et al. También estableció un relativamente complejo en el modelo de silico para predecir de plano de transporte linfático de compuestos lipófilos sobre la base de los descriptores moleculares 42. Estos modelos pueden proporcionar un enfoque útil para predecir el grado de transporte linfático de fármacos desconocidos. Será, sin embargo, se requerirá la validación de los modelos con una amplia gama de medicamentos y en los diferentes laboratorios para confirmar su exactitud y reproducibilidad. La canulación del conducto linfático mesentérico por lo tanto sigue siendo el único medio para examinar directamente el contenido de drenaje linfático del intestino delgado y la velocidad de tránsito de la compleja serie de factores (células, proteínas,péptidos, lípidos, drogas) en la linfa en una situación in vivo. Aquí se describe un protocolo para la canulación del conducto linfático y carótida arteria mesentérica que permite la recogida de la linfa mesentérica y la sangre sistémica de las ratas anestesiadas. Los datos representativos demuestran cómo el modelo puede ser utilizado para examinar los lípidos y el transporte del fármaco desde el intestino a través del sistema linfático mesentérico. Esto es seguido por una discusión de las dificultades que se pueden encontrar en el establecimiento del modelo y una guía para resolver problemas. Una vez establecido el modelo es una herramienta poderosa para investigar el transporte linfático intestinal.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Sterile saline</td>
			<td>Baxter healthcare</td>
			<td>AHB 1307</td>
			<td>Any brand can be used. Example here is Baxter 100 ml saline bags, box of 50</td>
		</tr>
		<tr >
			<td >70 % ethanol in water</td>
			<td>Any</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Chlorhexidine gluconate solution (Microshield 4)</td>
			<td>Livingstone International</td>
			<td>JJ60243L</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=JJ60243L</td>
		</tr>
		<tr >
			<td >Betadine solution</td>
			<td>Livingstone International</td>
			<td>BU0510</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=BU0510</td>
		</tr>
		<tr >
			<td >Ilium Ketamil (Ketamine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >KETA I 1</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Ilium Xylazil (Xylazine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >TRO-3828</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >ACP 10 Injection (Acepromazine 10 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >VTG-DACP010020</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Sodium pentobarbitone</td>
			<td>PROVET VICTORIA&#160;</td>
			<td>24529</td>
			<td>Any brand can be used. Example here is Lethabarb&#174; 325 mg/ml sodium pentobarbitone, Virbac Animal Health. http://www.provet.com.au</td>
		</tr>
		<tr >
			<td >Heparin (35000I.U. in 35 mL)</td>
			<td>Sigma Pharmaceuticals</td>
			<td>337220</td>
			<td>http://sigmaco.com.au/</td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate</td>
			<td >Sigma-Aldrich</td>
			<td >E1644</td>
			<td>Any brand can be used. Example here is disodium salt of EDTA from Sigma.&#160;</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.96 mm x i.d. 0.58 mm</td>
			<td>Microtube extensions</td>
			<td>PE8050</td>
			<td>Any brand can be used. Example here is PE tubing 0.8x0.5 mm, 30 m</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.8 mm x i.d. 0.5 mm</td>
			<td>Microtube extensions</td>
			<td>PE9658</td>
			<td>Any brand can be used. Example here is PE tubing 0.96x0.58 mm, 30 m</td>
		</tr>
		<tr >
			<td >Ruler</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Markers</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cigarette lighter</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cyanoacrylate glue</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >23 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN23GX0.75LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 23GX0.75inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT= 
			6&#38;search=DN23GX0.75LV</td>
		</tr>
		<tr >
			<td >25 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN25GX1.0LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 25GX1.0inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search= 
			DN25GX1.0LV</td>
		</tr>
		<tr >
			<td >1 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS01TA</td>
			<td>Any brand can be used. Example here is Terumo syringe 1 ml Slip Tuberculin 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS01TA</td>
		</tr>
		<tr >
			<td >10 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS10SA</td>
			<td>Any brand can be used. Example here is Terumo syringe 10 ml Slip 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS10SA</td>
		</tr>
		<tr >
			<td >Gauze swabs</td>
			<td>Livingstone International</td>
			<td>GSC075</td>
			<td>Any brand can be used and cut to required size. Example here is gauze swabs cotton filled 7.5x7.5 cm, 8 ply. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=GSC075</td>
		</tr>
		<tr >
			<td >Cotton buds</td>
			<td>Livingstone International</td>
			<td>CTAST075DP</td>
			<td>Any brand can be used. Example here is Livingstone cotton applicator plastic double tipped. 75MM. 100/PK. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=CTAST075DP</td>
		</tr>
		<tr >
			<td >Heating pad</td>
			<td>Ratek</td>
			<td>WT1</td>
			<td>Any brand that keeps temperature at 37C can be used. Example here is Ratek warming tray.</td>
		</tr>
		<tr >
			<td >Surgical light</td>
			<td>Harvard Apparatus</td>
			<td>72-0215 with 72-0267</td>
			<td>Any brand can be used. Example here is Harvard apparatus V-Lux 1000 Cold Light Source with Bifurcated Gooseneck Light Guide, Black, 4.7 mm fiber diameter (each arm). http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_50601_ 
			-1_HAI_ProductDetail and&#160; http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_35487_ 
			-1_HAI_ProductDetail___</td>
		</tr>
		<tr >
			<td >Surgical microscope</td>
			<td>Zeiss</td>
			<td>495005-0014-000</td>
			<td>Any brand can be used. Example here is Zeiss Stereomicroscope Stemi 2000-C with Stand S Double Spot and KL 300 LED. https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=e&#38;i=10143</td>
		</tr>
		<tr >
			<td >Silk suture</td>
			<td>Livingstone International</td>
			<td>DTSK163019F4</td>
			<td>Any brand can be used. Example here is *&#160; 
			Email this item to my friend 
			3/8 Circle Reverse Cut Silk Suture 3/0 Thread 19mm. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=DTSK163019F4</td>
		</tr>
		<tr >
			<td >Scalpel blades</td>
			<td >Fine Science Tools (FST)</td>
			<td >10020-00</td>
			<td>Any brand can be used. Example here is FST Scalpel Blade #20. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=191</td>
		</tr>
		<tr >
			<td >Scalpel handle</td>
			<td >Fine Science Tools (FST)</td>
			<td>10004-13</td>
			<td>Any brand can be used. Example here is FST Scalpel Handle #4. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=298&#38;CategoryId=51</td>
		</tr>
		<tr >
			<td >1 x Small surgical scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>14060-09</td>
			<td>Any brand can be used. Example here is FST Fine Scissors, 9 cm with 21 mm cutting edge, sharp, straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=40&#38;CategoryId=17</td>
		</tr>
		<tr >
			<td >2 x Forceps with serrated curved tip</td>
			<td >Fine Science Tools (FST)</td>
			<td >11001-13</td>
			<td>Any brand can be used. Example here is FST 13 cm standard pattern forceps with curved 2.8x1.4 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=405&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Iridectomy scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>15000-08</td>
			<td>Any brand can be used. Example here is FST Vannas Spring Scissors - 2.5mm Cutting Edge, Straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=17&#38;CategoryId=16&#160;</td>
		</tr>
		<tr >
			<td >1 x Forceps with straight serated tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11650-10</td>
			<td>Any brand can be used. Example here is FST Graefe 10 cm straight with serrated 1 x 0.99 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=390&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth sharp straight fine tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11251-10</td>
			<td>Any brand can be used. Example here is FST Dumont #5 forceps straight 11cm with 0.08 x 0.04mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=335&#38;CategoryId=29</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth fine curved forceps</td>
			<td >Fine Science Tools (FST)</td>
			<td>11063-07</td>
			<td>Any brand can be used. Example here is FST Delicate Forceps 9 cm with smooth 0.4 x 0.3mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=360</td>
		</tr>
		<tr >
			<td >2 x Hemostats</td>
			<td >Fine Science Tools (FST)</td>
			<td >13010-12</td>
			<td>Any brand can be used. Not all operators use the hemostats. Example here is FST 12 cm Micro-Mosquito Hemostats with 20 mm length x 1.3 mm width serrated, straight tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=377&#38;CategoryId=33</td>
		</tr>
		<tr >
			<td >1 x Suture needle holder</td>
			<td >Fine Science Tools (FST)</td>
			<td>12001-13</td>
			<td>Any brand can be used. Example here is FST 13cm Hasley Needle Holder with 16 mm length x 1.9 mm width tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=254&#38;CategoryId=70</td>
		</tr>
		<tr >
			<td >1 x Artery clamp</td>
			<td >Fine Science Tools (FST)</td>
			<td>18050-28</td>
			<td>Any brand can be used. Example here is FST Bulldog Serrefines straight, 28 mm long, 9x1.6 mm jaw dimension with medium clamp press. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=270&#38;CategoryId=82</td>
		</tr>
		<tr >
			<td >Oleic acid</td>
			<td >Sigma Aldrich</td>
			<td >O1008</td>
			<td>When required, any brand can be used. Example here is 99% pure oleic acid. http://www.sigmaaldrich.com/catalog/product/sial/o1008?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >14C-oleic acid</td>
			<td >Perkin&#160;</td>
			<td>NEC317050UC&#160;</td>
			<td>Any brand can be used. Example here is Oleic Acid, [1-14C]-, 50&#181;Ci (1.85MBq). http://www.perkinelmer.com/Catalog/Product/ID/NEC317050UC</td>
		</tr>
		<tr >
			<td >Sodium taurocholate</td>
			<td >Sigma Aldrich</td>
			<td >T4009</td>
			<td>Any brand can be used. Example here is taurocholic acid sodium salt hydrate &#8805;95% (TLC) . http://www.sigmaaldrich.com/catalog/product/sigma/t4009?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Halofantrine</td>
			<td >Glaxo Smith Kline</td>
			<td ></td>
			<td>Halofantrine was kindly provided as a gift from Glaxo Smith Kline</td>
		</tr>
		<tr >
			<td >Sodium phosphate monobasic</td>
			<td >Sigma Aldrich</td>
			<td >71507</td>
			<td >Any brand can be used. Example here is sodium phosphate monobasic monohydrate, BioXtra, for molecular biology, &#62;99.5%. http://www.sigmaaldrich.com/catalog/product/sigma/71643?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Sodium phosphate dibasic</td>
			<td >Sigma Aldrich</td>
			<td >71643</td>
			<td >Any brand can be used. Example here is sodium phosphate dibasic dihydrate, BioUltra, for molecular biology, &#62;99%. http://www.sigmaaldrich.com/catalog/product/sigma/71507?lang=en&#38;region=AU</td>
		</tr>
	</tbody>
</table>

the mesenteric lymph duct cannulated rat model: application to the assessment of intestinal lymphatic drug transport

El sistema linfático intestinal juega un papel clave en el transporte de líquidos, la absorción de lípidos y la función inmune. La linfa fluye directamente desde el intestino delgado a través de una serie de vasos linfáticos y los nodos que convergen en el conducto linfático mesentérico superior. La canulación del conducto linfático mesentérico por lo tanto permite la recogida de la linfa mesentérica que fluye desde el intestino. Linfático mesentérico consiste en una fracción celular de las células inmunes (99% de linfocitos), fracción acuosa (líquido, péptidos y proteínas tales como citocinas y hormonas intestinales) y la fracción de lipoproteínas (lípidos, moléculas lipófilas y apo-proteínas). Por consiguiente, el modelo de la canulación del conducto linfático mesentérico se puede utilizar para medir la concentración y la velocidad de transporte de una serie de factores del intestino a través del sistema linfático. Los cambios en estos factores en respuesta a diferentes retos (por ejemplo, las dietas, antígenos, fármacos) y en la enfermedad (por ejemplo, enfermedad inflamatoria del intestino, VIH, diabetes) puede también be determinada. Un área de expansión de interés es el papel del transporte linfático en la absorción de fármacos lipófilos administrados por vía oral y profármacos que se asocian con las vías de absorción de lípidos intestinales. Aquí se describe, en detalle, un modelo de rata canulado conducto linfático mesentérico, que permite la evaluación de la velocidad y extensión de los lípidos y el transporte del fármaco a través del sistema linfático durante varias horas después del parto intestinal. El método es fácilmente adaptable a la medición de otros parámetros en los ganglios. Proporcionamos una descripción detallada de las dificultades que se pueden encontrar al establecer este método quirúrgico complejo, así como los datos representativos de experimentos fallidos y exitosos para proveer instrucción sobre cómo confirmar el éxito experimental e interpretar los datos obtenidos.

Los resultados de un experimento representativo para cuantificar la medida acumulativa y la tasa de lípidos y el transporte del fármaco a través del sistema linfático intestinal después de la entrega utilizando el modelo de canulación linfático mesentérico se muestra en la Figura 4 y la Figura 5. En este experimento, 200 g de el modelo lipófilo halofantrina fármaco se administró en el duodeno de ratas durante 2 h en una formulación que contiene 40 mg de ácido oleico (incluy...

Watch this Scientific Journal Video about The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport at JoVE.com

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

La linfa mesentérica Conducto canulado Rata Modelo: Aplicación de la Evaluación de Intestinal Linfático Transporte de Drogas

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

the-mesenteric-lymph-duct-cannulated-rat-model-application-to

Research

JoVE Journal

Immunology and Infection

28.2K vistas.  Monash University (Parkville Campus). Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

Video: The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopia intravital de los ganglios linfáticos inguinales

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Investigación

Inmunología e Infección

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagen intravital del nodo linfático poplíteo ratón

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

No invasiva imágenes ópticas de la vasculatura linfática de un ratón

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Generación de nódulos linfáticos de grasa Pad quimeras para el Estudio del Ganglio Linfático estroma origen de célula

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Método del agitador magnético para la detección de<em&gt; Trichinella</em&gt; Las larvas en muestras de músculo

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

Cuantitativa de polimerasa análisis reacción-basado cadena de la Microbiota Intestinal murino después del tratamiento antibiótico Oral

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Infección crónica por salmonela fibrosis intestinal inducida

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Bloqueo del flujo linfático mediante la sutura de los vasos linfáticos aferentes en ratones

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

Evaluación Funcional De La Permeabilidad Intestinal Y La Migración Transepitelial Neutrófilo En Ratones Usando Un Modelo De Bucle Intestinal Estandarizado

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

Organoides intestinales caninos en un sistema de soporte permeable de doble cámara

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...